Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Enhancing motor functional recovery in spinal cord injury through pharmacological inhibition of Dickkopf-1 with BHQ880 antibody.

doi: 10.1016/j.biopha.2024.116792

Figure Lengend Snippet: Fig. 1. In vitro evaluation of the proper effect of the BHQ880 antibody over Dkk1 protein neutralization. (A) Representative images of B1a/7TGC cell reporter line activation when stimulated by the GSK-3 inhibitor CHIR99021 (positive control) or by increasing doses of recombinant mouse Wnt3a (mWnt3a), and data obtained from the evaluation of the percentage of GFP+ cells after 48 hours. *vs. Non-treated (NT); # vs. 0 ng/mL mWnt3a. (B) Representative images of B1a/7TGC cell reporter line inactivation when previously stimulated cells with 150 ng/mL of mWnt3a were turned off through the addition of increasing doses of recombinant rDkk1, and data obtained from the evaluation of the percentage of GFP+ cells after 48 hours. *vs. NT; # vs. 0 ng/mL rDkk1. (C) Representative images of the BHQ880 neutralizing effect over rDkk1 protein in the B1a/7TGC reporter cells stimulated with 150 ng/mL of mWnt3a, together with 250 ng/mL of rDkk1 and increasing doses of BHQ880 antibody, and data obtained from the evaluation of the percentage of GFP+ cells after 48 hours. *vs. NT; & vs. Wnt3a; # vs 0 ng/mL BHQ880. A one-way ANOVA followed by a Bonferroni post-hoc test was used to assess the existence of significant differences between groups. In all cases, scale bars = 50 μm and the data are presented as the mean + SEM. **p <0.01; ***p <0.001; #p <0.05; ##p <0.01; ###p <0.001; &&p <0.01.

Article Snippet: Finally, to validate the specificity of the Dkk1 primary antibody used in these experiments, we pre-adsorbed the antibody with its corresponding blocking peptide (Proteintech, AG15110) at a 20-fold weight/ weight excess following the protocol previously described [51], and we observed that pre-adsorption abolished Dkk1 immunostaining, thus confirming antibody specificity (Fig. S1).

Techniques: In Vitro, Neutralization, Activation Assay, Positive Control, Recombinant

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Enhancing motor functional recovery in spinal cord injury through pharmacological inhibition of Dickkopf-1 with BHQ880 antibody.

doi: 10.1016/j.biopha.2024.116792

Figure Lengend Snippet: Fig. 2. Protein expression of Dkk1 in the healthy and injured spinal cord. The figure shows representative images from the immunohistochemical evaluation of Dickkopf-1 (Dkk1) protein expression in the non-lesioned (NL) (n = 5) spinal cord (A) and after SCI at 24 hours post-injury (hpi) (n = 4), and 3 days post-injury (dpi), 7 dpi (n = 4), 14 dpi (n = 5), 28 dpi (n = 3), and 42 dpi (n = 5) at the lesion epicenter (B-G). Dashed line squares in A-G indicate the areas shown in the corresponding higher magnification images. Scale bars = 500 μm. Inserts scale bars = 50 μm. (H) Densitometric analysis of Dkk1 immunolabeling in the NL spinal cord and at the injury epicenter after SCI from 24 hpi to 42 dpi. A one-way ANOVA followed by a Bonferroni post-hoc test was used to assess the existence of significant differences between NL and the different times post-injury. In all cases, the data are presented as the mean + SEM. **p <0.01; ***p <0.001.

Article Snippet: Finally, to validate the specificity of the Dkk1 primary antibody used in these experiments, we pre-adsorbed the antibody with its corresponding blocking peptide (Proteintech, AG15110) at a 20-fold weight/ weight excess following the protocol previously described [51], and we observed that pre-adsorption abolished Dkk1 immunostaining, thus confirming antibody specificity (Fig. S1).

Techniques: Expressing, Immunohistochemical staining, Immunolabeling

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Enhancing motor functional recovery in spinal cord injury through pharmacological inhibition of Dickkopf-1 with BHQ880 antibody.

doi: 10.1016/j.biopha.2024.116792

Figure Lengend Snippet: Fig. 3. Analysis of the circulating levels of Dkk1 in Serum samples from non- lesioned (NL) and lesioned rats at 24 hours after damage (SCI). The figure shows data obtained from the quantitative analysis of serum circulating Dkk1 protein levels measured by ELISA in NL animals (n = 9) and injured animals 24 hours after SCI (n = 9). No significant differences were observed between groups. A two-tailed t test was used to determine the existence of significant differences between groups. The data represent the mean ± SEM.

Article Snippet: Finally, to validate the specificity of the Dkk1 primary antibody used in these experiments, we pre-adsorbed the antibody with its corresponding blocking peptide (Proteintech, AG15110) at a 20-fold weight/ weight excess following the protocol previously described [51], and we observed that pre-adsorption abolished Dkk1 immunostaining, thus confirming antibody specificity (Fig. S1).

Techniques: Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal:

Article Title:

doi:

Figure Lengend Snippet: Effect of CT on the expression of matrix degrading enzymes in IL-1β-injured rat chondrocytes. (A) mRNA and (B) protein levels of MMP-13, MMP-3, MMP-9, ADAMTS4 and ADAMTS5 were assessed using reverse transcription-quantitative PCR and western blot analysis. **P<0.01 vs. Normal group; ## P<0.01 vs. IL-1β group; && P<0.01 vs. IL-1β + CT (50 nM); $$ P<0.01 vs. IL-1β + IWR-1-endo. CT, calcitonin; IL-1β, interleukin-1β; MMP, matrix metallopeptidase; ADAMTS, ADAM metallopeptidase with thrombospondin type 1; Dkk-1, anti-dickkopf-1.

Article Snippet: After blocking with 5% non-fat milk at 4°C overnight, PVDF membranes were subsequently incubated with the following primary antibodies at 4°C overnight: Anti-dickkopf-1 (Dkk-1; cat. no. ab109416; 1:1,000; Abcam), anti-β-catenin (cat. no. ab32572; 1:1,500; Abcam), anti-MMP13 (cat. no. ab39012; 1:3,000; Abcam), anti-ADAMTS4 (cat. no. ab185722; 1:500; Abcam), anti-Bax (cat. no. 5023; 1:1,000; Cell Signaling Technology Inc.), anti-Bcl-2 (cat. no. 15071; 1:1,000; Cell Signaling Technology, Inc.), anti-cleaved-caspase 3 (cat. no. 9664; 1:1,000; Cell Signaling Technology, Inc.), anti-MMP-3 (cat. no. ab53015; 1:1,000; Abcam), anti-MMP-9 (cat. no. ab38898; 1:500; Abcam), anti-ADAMTS5 (cat. no. ab41037; 1:1,000; Abcam) and anti-GAPDH (cat. no. 5174; 1:2,000; Cell Signaling Technology, Inc.).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Dickkopf-1 Acts as a Profibrotic Mediator in Progressive Chronic Kidney Disease

doi: 10.3390/ijms24087679

Figure Lengend Snippet: Elevated levels of serum DKK1 are associated with fast progression to ESRD in patients with CKD stages 4–5. ( A ) Serum DKK1 levels in normal controls (n = 15) and in patients with CKD stages 4–5 (n = 50). Data are presented as mean ± SD. ( B ) Western blot analysis for the expression of DKK1, fibronectin (FBN) and TGF-β1 in renal tissues from non-CKD and CKD subjects. Quantitative data are expressed as mean ± SD. * p < 0.05 versus control subjects (n = 6). ( C ) Chromogenic IHC staining for DKK1, FBN and TGF-β1 in renal glomeruli of representative non-CKD subjects (#1 and #2) and CKD subjects (#1 and #2). Scale bars: 20 μm. ( D ) Chromogenic IHC staining for DKK1, FBN and TGF-β1 in renal tubules of representative non-CKD subjects (#1 and #2) and CKD subjects (#1 and #2). Scale bars: 20 μm. ( E ) One minus dialysis-free survival analysis of CKD 4–5 patients with high serum levels of DKK1 (≥1526.4 pg/mL; n = 22) or low serum levels of DKK1 (<1526.4 pg/mL; n = 28) in an 8-year follow-up. ( F ) Stratification by 3-year follow-up in CKD 4–5 patients with low or high serum levels of DKK1.

Article Snippet: For immunohistochemistry, human kidney sections were stained with the primary antibodies against DKK1 (sc-25516; Santa Cruz, Dallas, TX, USA), fibronectin (ab45688; Abcam, Cambridge, UK) or TGF-β1 (BS1361; Bioworld Technology, Dublin, OH, USA), whereas rat kidney sections were stained with antibodies against DKK1 (sc-25516; Santa Cruz, Dallas, TX, USA), TGF-β1 (E11264; Spring Bioscience, Pleasanton, CA, USA), collagen IV (BSB5355; BioSB, Santa Barbara, CA, USA), fibronectin (BS1644; Bioworld Technology, Dublin, OH, USA) or IL-1β (sc-7884; Santa Cruz, Dallas, TX, USA).

Techniques: Western Blot, Expressing, Control, Immunohistochemistry

Journal: International Journal of Molecular Sciences

Article Title: Dickkopf-1 Acts as a Profibrotic Mediator in Progressive Chronic Kidney Disease

doi: 10.3390/ijms24087679

Figure Lengend Snippet: Baseline characteristics of advanced CKD patients stratified by the optimal cutoff level of serum DKK1 (1526.4 pg/mL).

Article Snippet: For immunohistochemistry, human kidney sections were stained with the primary antibodies against DKK1 (sc-25516; Santa Cruz, Dallas, TX, USA), fibronectin (ab45688; Abcam, Cambridge, UK) or TGF-β1 (BS1361; Bioworld Technology, Dublin, OH, USA), whereas rat kidney sections were stained with antibodies against DKK1 (sc-25516; Santa Cruz, Dallas, TX, USA), TGF-β1 (E11264; Spring Bioscience, Pleasanton, CA, USA), collagen IV (BSB5355; BioSB, Santa Barbara, CA, USA), fibronectin (BS1644; Bioworld Technology, Dublin, OH, USA) or IL-1β (sc-7884; Santa Cruz, Dallas, TX, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Dickkopf-1 Acts as a Profibrotic Mediator in Progressive Chronic Kidney Disease

doi: 10.3390/ijms24087679

Figure Lengend Snippet: Association of serum DKK1 level (per 100 pg/mL increase) with the risk of outcomes (n = 50).

Article Snippet: For immunohistochemistry, human kidney sections were stained with the primary antibodies against DKK1 (sc-25516; Santa Cruz, Dallas, TX, USA), fibronectin (ab45688; Abcam, Cambridge, UK) or TGF-β1 (BS1361; Bioworld Technology, Dublin, OH, USA), whereas rat kidney sections were stained with antibodies against DKK1 (sc-25516; Santa Cruz, Dallas, TX, USA), TGF-β1 (E11264; Spring Bioscience, Pleasanton, CA, USA), collagen IV (BSB5355; BioSB, Santa Barbara, CA, USA), fibronectin (BS1644; Bioworld Technology, Dublin, OH, USA) or IL-1β (sc-7884; Santa Cruz, Dallas, TX, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Dickkopf-1 Acts as a Profibrotic Mediator in Progressive Chronic Kidney Disease

doi: 10.3390/ijms24087679

Figure Lengend Snippet: Treatment of 5/6-nephrectomized (5/6 Nx) rats with the DKK1-antisense (DKK1-AS) oligonucleotide alleviates CKD-associated phenotypes. Body weight ( A ), blood urea nitrogen (BUN) level ( B ), serum creatinine (Cr) level ( C ), urine albumin/creatinine ratio (ACR) ( D ), urine total protein/creatinine ratio (TP/Cr) ( E ) and serum DKK1 level ( F ) were analyzed in the sham-operated rats (NC) and in the 5/6 Nx rats that were left untreated or treated with the DKK1-sense (DKK1-S) or DKK1-antisense (DKK1-AS) oligonucleotide. Data are expressed as mean ± SD (n = 6 for each group). * p < 0.01 versus the sham group, # p < 0.01 versus the untreated 5/6 Nx group. ( G ) Representative images of PAS staining and IHC staining for DKK1 in renal glomeruli of the indicated treated groups. Scale bars: 50 μm. Intensities of PAS staining and IHC staining for DKK1 in kidney sections from different groups were determined by quantitative integrated optical density (IOD) analysis and plotted in panels ( i ) and ( ii ), respectively. Data are represented as mean ± SD (n = 6 for each group). * p < 0.05 versus the sham group, # p < 0.05 versus the untreated 5/6 Nx group.

Article Snippet: For immunohistochemistry, human kidney sections were stained with the primary antibodies against DKK1 (sc-25516; Santa Cruz, Dallas, TX, USA), fibronectin (ab45688; Abcam, Cambridge, UK) or TGF-β1 (BS1361; Bioworld Technology, Dublin, OH, USA), whereas rat kidney sections were stained with antibodies against DKK1 (sc-25516; Santa Cruz, Dallas, TX, USA), TGF-β1 (E11264; Spring Bioscience, Pleasanton, CA, USA), collagen IV (BSB5355; BioSB, Santa Barbara, CA, USA), fibronectin (BS1644; Bioworld Technology, Dublin, OH, USA) or IL-1β (sc-7884; Santa Cruz, Dallas, TX, USA).

Techniques: Staining, Immunohistochemistry

Journal: International Journal of Molecular Sciences

Article Title: Dickkopf-1 Acts as a Profibrotic Mediator in Progressive Chronic Kidney Disease

doi: 10.3390/ijms24087679

Figure Lengend Snippet: Evaluation of the expression levels of profibrotic and proinflammatory factors in renal tissues of the sham-operated rats and the 5/6 Nx rats left untreated or treated with DKK1-S or DKK-AS. ( A ) Representative photographs of renal sections before and after capturing glomerular compartments using laser capture microdissection (LCM). Scale bars: 100 μm. ( B ) Quantitative RT-PCR analysis of DKK1, TGF-β1 and fibronectin (FBN) mRNAs in isolated glomeruli microdissected from kidney sections of different groups. Quantitative data are represented as mean ± SD (n = 6 for each group). * p < 0.05 versus the sham group, # p < 0.05 versus the untreated 5/6 Nx group. ( C ) Representative images of Western blot analysis for the expression of DKK1, β-catenin, TGF-β1 and FBN in renal tissues of different groups. ( D ) Quantitative analysis of Western blots for DKK1, β-catenin, TGF-β1 and FBN in renal tissues of different groups. Data are plotted as mean ± SD (n = 6 for each group). * p < 0.05 versus the sham group, # p < 0.05 versus the untreated 5/6 Nx group. ( E ) IHC staining for TGF-β1, collagen IV (COL-IV), fibronectin (FBN) and IL-1β in renal glomeruli of different treated groups. Scale bars: 50 μm. Bottom panels show quantitative IOD analysis of the intensities of TGF-β1 ( i ), COL-IV ( ii ), FBN ( iii ) and IL-1β ( iv ) in renal glomeruli of different groups (n = 6 for each group). Data are represented as mean ± SD. * p < 0.05 versus the sham group, # p < 0.05 versus the untreated 5/6 Nx group.

Article Snippet: For immunohistochemistry, human kidney sections were stained with the primary antibodies against DKK1 (sc-25516; Santa Cruz, Dallas, TX, USA), fibronectin (ab45688; Abcam, Cambridge, UK) or TGF-β1 (BS1361; Bioworld Technology, Dublin, OH, USA), whereas rat kidney sections were stained with antibodies against DKK1 (sc-25516; Santa Cruz, Dallas, TX, USA), TGF-β1 (E11264; Spring Bioscience, Pleasanton, CA, USA), collagen IV (BSB5355; BioSB, Santa Barbara, CA, USA), fibronectin (BS1644; Bioworld Technology, Dublin, OH, USA) or IL-1β (sc-7884; Santa Cruz, Dallas, TX, USA).

Techniques: Expressing, Laser Capture Microdissection, Quantitative RT-PCR, Isolation, Western Blot, Immunohistochemistry

Journal: International Journal of Molecular Sciences

Article Title: Dickkopf-1 Acts as a Profibrotic Mediator in Progressive Chronic Kidney Disease

doi: 10.3390/ijms24087679

Figure Lengend Snippet: The renal expression of DKK1 positively correlates with accumulation of β-catenin in 5/6 Nx rats. ( A ) Representative images of Western blot analysis for the expression of DKK1 and β-catenin in renal tissues of the sham group and the 5/6 Nx groups that were untreated or treated with DKK1-S or DKK1-AS. ( B ) Quantitative analysis of Western blots for β-catenin in renal tissues of different groups (n = 6 for each group). * p < 0.05 versus the sham group, # p < 0.05 versus the untreated 5/6 Nx group. ( C ) Immunofluorescence analysis of β-catenin in kidney sections from the sham-operated rats and the 5/6 Nx rats left untreated or treated with DKK1-S or DKK-AS. Scale bars: 100 μm. ( D ) Immunofluorescence images of renal glomeruli stained for β-catenin in different treated groups. Scale bars: 50 μm. ( E ) Quantification of relative fluorescence intensities for β-catenin in renal glomeruli of different groups (n = 6 for each group). * p < 0.05 versus the sham group, # p < 0.05 versus the untreated 5/6 Nx group.

Article Snippet: For immunohistochemistry, human kidney sections were stained with the primary antibodies against DKK1 (sc-25516; Santa Cruz, Dallas, TX, USA), fibronectin (ab45688; Abcam, Cambridge, UK) or TGF-β1 (BS1361; Bioworld Technology, Dublin, OH, USA), whereas rat kidney sections were stained with antibodies against DKK1 (sc-25516; Santa Cruz, Dallas, TX, USA), TGF-β1 (E11264; Spring Bioscience, Pleasanton, CA, USA), collagen IV (BSB5355; BioSB, Santa Barbara, CA, USA), fibronectin (BS1644; Bioworld Technology, Dublin, OH, USA) or IL-1β (sc-7884; Santa Cruz, Dallas, TX, USA).

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Dickkopf-1 Acts as a Profibrotic Mediator in Progressive Chronic Kidney Disease

doi: 10.3390/ijms24087679

Figure Lengend Snippet: Uremic serum promotes fibrogenesis of renal mesangial cells through the DKK1-mediated signaling pathway. ( A ) Western blot analysis of fibronectin (FBN), α-smooth muscle actin (α-SMA), and collagen IV (COL-IV) expressed in mouse mesangial cells that were cultured in media with increasing concentrations (5, 10 and 20%) of healthy control serum or uremic serum. The healthy serum and uremic serum samples were pooled at equal volumes from blood samples of five healthy control subjects and five uremic patients, respectively. Quantitative data from Western blots are plotted as mean ± SD (n = 3). * p < 0.05 versus the healthy controls at the corresponding serum concentrations. ( B ) Western blot analysis of FBN, α-SMA and COL-IV expressed in mouse mesangial cells cultured in media with 20% healthy control serum or uremic serum for 24, 48 and 72 h. Quantitative data from Western blots are plotted as mean ± SD (n = 3). * p < 0.05 versus the healthy controls at the corresponding time points. ( C ) Serum DKK1 levels of five healthy control subjects and five uremic patients included in the in vitro experiments. ( D ) Effects of increasing amounts of anti-DKK1 neutralizing antibody (16.5, 66 and 200 ng/mL) on the expression of FBN, α-SMA and COL-IV in mesangial cells cultured in media with 20% healthy control serum or uremic serum. Quantitative data from Western blots are plotted as mean ± SD (n = 3). * p < 0.05 versus the healthy controls, # p < 0.05 versus the untreated uremic serum group. ( E ) Effects of increasing amounts of an IgG control and anti-DKK1 antibody on the expression of profibrotic proteins in mesangial cells cultured in media with 20% uremic serum. Quantitative data from Western blots are plotted as mean ± SD (n = 3). # p < 0.05 versus the untreated uremic serum group.

Article Snippet: For immunohistochemistry, human kidney sections were stained with the primary antibodies against DKK1 (sc-25516; Santa Cruz, Dallas, TX, USA), fibronectin (ab45688; Abcam, Cambridge, UK) or TGF-β1 (BS1361; Bioworld Technology, Dublin, OH, USA), whereas rat kidney sections were stained with antibodies against DKK1 (sc-25516; Santa Cruz, Dallas, TX, USA), TGF-β1 (E11264; Spring Bioscience, Pleasanton, CA, USA), collagen IV (BSB5355; BioSB, Santa Barbara, CA, USA), fibronectin (BS1644; Bioworld Technology, Dublin, OH, USA) or IL-1β (sc-7884; Santa Cruz, Dallas, TX, USA).

Techniques: Western Blot, Cell Culture, Control, In Vitro, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Dickkopf-1 Acts as a Profibrotic Mediator in Progressive Chronic Kidney Disease

doi: 10.3390/ijms24087679

Figure Lengend Snippet: DKK1 acts as a profibrotic mediator in mouse mesangial cells. ( A ) Effects of increasing amounts of recombinant human DKK1 (100, 200 and 400 ng/mL) on the expression of FBN, COL-IV, TGF-β1, α-SMA, DKK1 and β-catenin in mouse mesangial cells. The quantitative results from Western blots are plotted as mean ± SD (n = 3). * p < 0.05 versus the untreated control. ( B ) Effects of increasing amounts of recombinant mouse DKK1 (100, 200 and 400 ng/mL) on the expression of the indicated proteins in mouse mesangial cells. The quantitative data from Western blots are plotted as mean ± SD (n = 3). * p < 0.05 versus the untreated control. ( C ) Quantitative RT-PCR analysis of FBN, TGF-β1 and DKK1 mRNAs expressed in mouse mesangial cells that were left untreated or treated with increasing amounts of recombinant human DKK1 (100, 200 and 400 ng/mL). * p < 0.05 versus the untreated control (n = 3). ( D ) Quantitative RT-PCR analysis of FBN, TGF-β1 and DKK1 mRNAs expressed in mouse mesangial cells that were left untreated or treated with increasing amounts of recombinant mouse DKK1 (100, 200 and 400 ng/mL). * p < 0.05 versus the untreated control (n = 3).

Article Snippet: For immunohistochemistry, human kidney sections were stained with the primary antibodies against DKK1 (sc-25516; Santa Cruz, Dallas, TX, USA), fibronectin (ab45688; Abcam, Cambridge, UK) or TGF-β1 (BS1361; Bioworld Technology, Dublin, OH, USA), whereas rat kidney sections were stained with antibodies against DKK1 (sc-25516; Santa Cruz, Dallas, TX, USA), TGF-β1 (E11264; Spring Bioscience, Pleasanton, CA, USA), collagen IV (BSB5355; BioSB, Santa Barbara, CA, USA), fibronectin (BS1644; Bioworld Technology, Dublin, OH, USA) or IL-1β (sc-7884; Santa Cruz, Dallas, TX, USA).

Techniques: Recombinant, Expressing, Western Blot, Control, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Dickkopf-1 Acts as a Profibrotic Mediator in Progressive Chronic Kidney Disease

doi: 10.3390/ijms24087679

Figure Lengend Snippet: Proposed model of DKK1-mediated renal fibrosis. During renal injury, DKK1 can be initially activated by the Wnt/β-catenin signaling pathway. Subsequently, DKK1 may autoregulate its own expression to further increase its levels. The DKK1 autoregulation may be unrelated to the Wnt/β-catenin signaling pathway. Increased expression of DKK1 is involved in renal fibrosis and thus promotes CKD progression. Under the circumstances of renal injury, DKK1 is no longer acting as a Wnt/β-catenin antagonist (via interaction with LRP5/6). Conversely, DKK1 may activate the Wnt/β-catenin signaling by some unknown mechanisms in renal cells or in infiltrating immune cells.

Article Snippet: For immunohistochemistry, human kidney sections were stained with the primary antibodies against DKK1 (sc-25516; Santa Cruz, Dallas, TX, USA), fibronectin (ab45688; Abcam, Cambridge, UK) or TGF-β1 (BS1361; Bioworld Technology, Dublin, OH, USA), whereas rat kidney sections were stained with antibodies against DKK1 (sc-25516; Santa Cruz, Dallas, TX, USA), TGF-β1 (E11264; Spring Bioscience, Pleasanton, CA, USA), collagen IV (BSB5355; BioSB, Santa Barbara, CA, USA), fibronectin (BS1644; Bioworld Technology, Dublin, OH, USA) or IL-1β (sc-7884; Santa Cruz, Dallas, TX, USA).

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Double Knockdown of the Androgen Receptor Target Genes DKK1 and SFRP1 Does Not Potentiate the Hair Growth-Promoting Effect of SFRP1 Silencing in Healthy Human Hair Follicles Ex Vivo

doi: 10.3390/ijms27041815

Figure Lengend Snippet: AR SNA efficiently downregulates AR mRNA and protein, as well as AR target gene expression in healthy human HFs. ( a ) Pooled quantification of AR mRNA expression assessed by qRT-PCR in healthy human HFs treated with 10 µM, 25 µM or 50 µM control or 10 µM, 25 µM or 50 µM AR SNA for 6 days, n = 4 independent donors. ( b , c ) Quantification ( b ) and representative images ( c ) of AR protein expression assessed by immunofluorescence staining in healthy human VI HFs treated with 10 µM control or 10 µM AR SNA for 6 days, in n = 7–8 HFs from 2 independent donors. Scale bars = 50 µM, in the original images and 25 µM in the zoom-ins. ( d ) Quantification of DKK1 and SFRP1 mRNA expression assessed by qRT-PCR in healthy human HFs treated with 10 µM control or 10 µM AR SNA for 6 days, n = 3 technical replicates from 1 donor. Dots of different shapes represent different donors. All data are presented as mean ± SEM. D’Agostino & Pearson normality test followed by Mann–Whitney test, * p < 0.05, ** p < 0.01. AR: androgen receptor, DKK1: dickkopf 1, DP: dermal papilla, DPst: dermal papilla stalk, DC: dermal cup, gHM: germinative hair matrix, HF: hair follicle, ORS: outer root sheath, pcHM: pre-cortical hair matrix, SFRP1: secreted frizzled receptor 1.

Article Snippet: Sections were then incubated overnight at 4 °C with the primary antibody against DKK1 (1:200; monoclonal mouse anti-DKK1, clone 2 A5, Novus Biologicals).

Techniques: Targeted Gene Expression, Expressing, Quantitative RT-PCR, Control, Immunofluorescence, Staining, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Double Knockdown of the Androgen Receptor Target Genes DKK1 and SFRP1 Does Not Potentiate the Hair Growth-Promoting Effect of SFRP1 Silencing in Healthy Human Hair Follicles Ex Vivo

doi: 10.3390/ijms27041815

Figure Lengend Snippet: Reciprocal modulation of DKK1 and SFRP1 after siRNA in HEK293 cells and DP fibroblasts in vitro and healthy human HFs ex vivo. ( a ) Quantification of DKK1 and SFRP1 mRNA expression assessed by qRT-PCR in HEK293 cells treated with 25 nM DKK1 or SFRP1 siRNA or 25 nM non-template control (NTC) siRNA for 48 h, n = 3 independent experiments, ( b ) or in human DP fibroblasts treated with 25 nM NTC siRNA, DKK1 siRNA or SFRP1 siRNA for 48 h, n = 2 technical replicates from 1 donor. ( c ) Quantification of DKK1 and SFRP1 mRNA expression in healthy human HFs, treated with 10 µM NTC siRNA, DKK1 siRNA or SFRP1 siRNA for 6 days, n = 4 independent donors. ( d ) Quantification of DKK1 and SFRP1 secretion from healthy human HFs, treated with each 10 µM NTC siRNA, DKK1 siRNA or SFRP1 siRNA. Medium was collected after 4 days of culture and following termination of the culture. n = 4 independent donors. All data are presented as mean ± SEM. Kruskal–Wallis test followed by Dunn’s multiple comparison test, # p < 0.05, or Mann–Whitney test, * p < 0.05. Dots of different shapes indicate different donors (filled dots) or experiments (empty dots). Data points reflecting the female donor are indicated by circles in c and d. DKK1: dickkopf 1, DP: dermal papilla, NTC: non template control, SFRP1: secreted frizzled receptor 1.

Article Snippet: Sections were then incubated overnight at 4 °C with the primary antibody against DKK1 (1:200; monoclonal mouse anti-DKK1, clone 2 A5, Novus Biologicals).

Techniques: In Vitro, Ex Vivo, Expressing, Quantitative RT-PCR, Control, Comparison, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Double Knockdown of the Androgen Receptor Target Genes DKK1 and SFRP1 Does Not Potentiate the Hair Growth-Promoting Effect of SFRP1 Silencing in Healthy Human Hair Follicles Ex Vivo

doi: 10.3390/ijms27041815

Figure Lengend Snippet: Silencing of SFRP1 prolongs anagen in healthy human HFs. Healthy human HFs were treated with 10 µM NTC siRNA, DKK1 siRNA, or SFRP1 siRNA for 5–8 days ex vivo. ( a ) Quantification of hair shaft production on days 1, 4, 5, 6, and 8 in culture with n = 24 (D1, D4), n = 6 (D5), n = 12–13 (D6), and n = 6 (D8) HFs from 1–4 independent donors. ( b ) Representative images of Ki-67/TUNEL immunofluorescence and Masson–Fontana histochemistry. ( c ) Quantitative analysis of hair cycle staging, n = 4 donors. ( d ) Quantitative analysis of the hair cycle score. Arbitrary units (au) were assigned to anagen (100), early catagen (200), mid catagen (300), and dystrophic (500) HFs. n = 23–24 HFs from 4 independent donors. ( e , f ) Quantitative analysis of hair matrix keratinocyte proliferation ( e ) and apoptosis ( f ). n = 23–24 HFs from 4 independent donors. All data are presented as mean ± SEM. Mann–Whitney test or unpaired t-test, * p < 0.05. Dots of different shapes indicate different donors. Data points reflecting the female donor are indicated by circles. DKK1: dickkopf 1, DP: dermal papilla, gHM: germinative hair matrix, HF: hair follicle, pcHM: pre-cortical hair matrix, SFRP1: secreted frizzled receptor 1.

Article Snippet: Sections were then incubated overnight at 4 °C with the primary antibody against DKK1 (1:200; monoclonal mouse anti-DKK1, clone 2 A5, Novus Biologicals).

Techniques: Ex Vivo, TUNEL Assay, Immunofluorescence, MANN-WHITNEY